Advanced stages of cancer represent a challenging therapeutic medical problem with a poor prognosis for the patient. These patients often require multiple medical treatments and interventions and in most cases treatment is purely empirical. As treatment costs rise with novel therapies, the need for reliable diagnostic tools to guide treatment decisions is paramount. As cancer is the second leading cause of death in the western world and in aging societies cancer becomes more prevalent, vast amount of efforts and financial resources are being invested in the development of novel therapeutical and diagnostic approaches. However, the essential step for the multitude of available new therapies is the efficient selection of patients for adequate cancer therapy. Currently, most of the currently known markers of cancer therapy efficacy are poorly reliable.
Standard classification for malignant tumors relies on the TNM system. The TNM (for “Tumor-Node-Metastasis”) classification system uses the size of the tumor, the presence or absence of tumor in regional lymph nodes, and the presence or absence of distant metastases, to assign a stage to the tumor (AJCC Cancer Staging Manual, Lippincott, 5thedition, pp. 171-180, 1997). Using this system, the assigned stage forms the basis for selection of appropriate therapy and also for prognostic purposes. Specifically, in colorectal cancers, the TNM system allows the distinction between (T) the degree of invasion of the intestinal wall, ranging from T0 to T4, (N) the degree of lymph node involvement, ranging from N0 to N3 and (M) the degree of metastasis, ranging from M0 to M1.
A meta-score for colorectal cancers is the Duke's classification. Duke's classification allows the distinction between at least four main tumor stages, respectively (A) tumor confined to the bowel wall, (B) tumor extending across the bowel wall, (C) involvement of regional nodes and (D) occurrence of distant metastases. A similar system is the staging system of the UICC, ranging from stage I (local tumor without metastases) to IV (tumor with distant organ metastases). These meta-scores are used to select patients for treatment options. UICC stage III patients usually receive chemotherapy after complete resection of the tumor tissue, while UICC stage IV usually receive palliative chemotherapy. It is still a controversy, whether UICC stage II patients require an adjuvant chemotherapy.
The above clinical classifications are useful in the clinical situation, but are completely imperfect in predicting the outcome of the intended therapy.
A similar situation can be found in other tumor entities. In breast cancer, expression of the protein HER2/neu by the tumor and other endocrine receptors is used to select patients for therapy strategies. Despite these selections, the variability in response to therapy is still big. Newer treatments with targeted drugs like monocolonal antibodies also give rise to some stratification of the patients. Modern chemotherapy regimens including monoclonal antibodies lead to objective response rates of around 50% in colorectal cancer patients, while almost half of the patients experience treatment related side effects without any clinical benefit. K-ras mutation is the only (negative) predictive marker for response to EGFR-targeting antibody treatment (Benvenuti et al. 2007, Cancer Res 67: 2643-2648) (Moroni, M et al. 2005, Lancet Oncol 6: 279-286). So far, no biomarkers are available that help to select patients that are likely to respond to chemotherapy.
Instead of conventional clinical staging, it has been proposed to use a large number of biological markers, including genes and proteins, that would be potentially useful for the diagnosis or the prognosis of a wide variety of cancers. Prognostic prediction and prediction of therapeutic efficacy are however not directly linked. Patients can have a response to a treatment but still have a shorter survival time then patients with no response to treatment (Farmer et al. 2009, Nat Med 15: 68-74).
It has not been proposed so far, that the presence of, or the expression level of, various biological markers of the host immune response in conjunction with the occurrence of a cancer at a given stage of cancer development was associated with the general response to treatment (i.e. treatment efficacy). For immunotherapeutic strategies, this association of a pre-existing immune response with a better therapeutic efficacy was assumed (see below). For chemotherapy, radiation therapy and other forms of drug intervention, this association has not been shown.
Nistico et al. (1999, Int. J. Cancer, Vol. 84: 598-603) assumed the existence of a spontaneous immune response against the erbB-2 oncogene product in HLA-A2-positive breast cancer patients. The impact of this immune response was thought to be dependent on tumor HLA-class-I molecule expression and on CD3+-T-lymphocyte localization, i.e. in intratumoral (IT) or peritumoral (PT) tissue. The authors reasoned, that these results could lead to the identification of new parameters that might be useful for defining more specific and more effective immunotherapeutic strategies against breast cancer.
A more elaborate approach was performed by Philips et al. (2004, British Journal of Surgery, Vol. 91: 469-475). They showed that tumour-infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic, by assaying both (i) the CD8/CD3 mRNA ratios and (ii) the CD3, CD4, CD8, cytokine IL-2Ra and Granzyme B protein production in the tumor tissue. There was however no significant correlation between mRNA copy numbers as T cell markers and immunohistochemical counts. Moreover there was no correlation with therapy response.
Maki et al. (2004, J. Gastroenterology and Hepatology, Vol. 19: 1348-1356) showed an impaired cellular immune system response in hepatocellular carcinoma-bearing patients. Decreased CD3[zeta] and CD28 protein expression by T cells was found in these patients, as well as an increased caspase-3 activity in CD28 down-modulated T cells. This suggests the occurrence of T cell apoptosis in HCC patients. Decreased expression of CD3[zeta] in T cells infiltrating cervical carcinoma was also reported by Grujil et al. (1999, British Journal of Cancer, Vol. 79: 1127-1132). These authors suggested that, in order for vaccination strategies to be successful, it might be essential to first identify and counteract mechanisms leading to this loss of CD3[zeta].
Ishigami et al. (2002, Cancer, Vol. 94 (5): 1437-1442) showed that reduced CD3-[zeta] expression is negatively correlated with lymph node involvement, depth of invasion, and clinical stage of gastric carcinoma. A reduced CD3-[zeta] expression correlates with a reduced 5-year survival rate of the patients, but only for patients which were diagnosed as “Stage IV” of gastric carcinoma. No information on treatment or association with treatment outcome is made.
An altered immune response in cancer patients was also found through the assessment of the expression of CD3, CD4, CD8 and Fas Ligand proteins on tumor-infiltrating lymphocytes (TILs) in head and neck cancer (Reichert et al. (2002, Clinical Cancer Research, Vol. 8: 3137-3145). This was also reported by Prado-Garcia et al. (2005, Lung Cancer, Vol. 47: 361-371), investigating the evasion mechanisms of lung adenocarcinoma by measuring the percentages of CD3+, CD4+ and CD8+ cells in peripheral blood and pleural effusion, and further CD27, CD28, CD45R0, CD45RA, granzyme A, Fas and perforin protein expression in the CD8+ T cell subsets. The characterization of these alterations that enable adenocarcinoma cells to inhibit CD8+ T cells in the initiation, growth and invasion processes of lung carcinoma, was proposed to allow the development of improved treatments for lung malignancies. Similar observations were made by Kuss et al. (2003, British Journal of Cancer, Vol. 88: 223-230)
Diederichsen et al. (2003, Cancer Immunol, Immunother., Vol. 52: 423-428) showed that colorectal patients with low CD4+/CD8+ ratios in TILs had a better clinical course, with significantly higher 5-year survival, independent of the Dukes stage and age.
Valmori et al. (2002, Cancer Research, Vol. 62:1743-1750) showed that the presence of a CD45RA+CCR7-CD8+ PBL T cell subset is associated with cytolytic activity in melanoma patients. These observations suggested an improved anti-tumor vaccination via the stimulation of such an effector immune response early in the course of the disease. The authors hypothesize, that such a response might be effective to eradicate minimal residual disease and prevent relapses.
Oshokiri et al. (2003, Journal of Surgical Oncology, Vol. 84: 224-228) reported a statistically significant association between the infiltration of cancer cell nests by CD8+ T cells and the survival in patients with extrahepatic bile duct carcinoma (EBDC). These authors showed that the level of CD8+ T cell infiltration correlated well with the conventional pTNM clinicopathological classification and that the infiltrate density was reliable for predicting the survival of patients with EBDC. Response to chemotherapy and association of the infiltrate density in this cancer entity was not investigated.
Menon et al. (2002, Lab Invest. 82, 1725-33) showed that the down-regulation of HLA-A expression correlates with a better prognosis in colorectal cancer patients. HLA molecules have a fundamental role in distinguishing “self versus” “not-self” (or “altered self”) for the immune system. Furthermore Menon et al. (2004, Lab Invest. 84, 493-501) conducted a detailed immunohistochemical analysis to corroborate the association between immune system and prognosis in colorectal cancer.
Furthermore, Zhang et al. (2003, New England Journal of Medicine, Vol. 348(3) 203-213) showed, that the presence or absence of intratumoral T cells correlates with the clinical outcome of advanced ovarian carcinoma after debulking and adjuvant chemotherapy. They however did not associate the outcome with the administered chemotherapy and thus no conclusion on the relation between infiltrate density and chemotherapy outcome was stated. The results were obtained through immunostaining assays of tumor cryosections with monoclonal antibodies against CD3, CD4, CD8, CD83, CD45, CD45R0, CD19, CD57 and CD11c, as well as through flow cytometry of cells from fresh tumor samples using monoclonal antibodies against HLADR, CD3, CD4, CD8, CD16, CD19, CD45, IgG1 and IgG2a. These authors had detected the presence or absence of CD3+ tumor-infiltrating T cells within tumor-cell islets and in peritumoral stroma. These authors reported that patients whose tumors contained higher numbers of T cells had both a median duration of (i) progression-free survival and (ii) overall survival which was statistically higher than patients whose tumors did not contain T cells. These authors suggested to further validate the use of detection of intratumoral T cells in the classification and treatment of patients with ovarian carcinoma.
Galon et al. (2006, Science, vol. 313, 1960-1964) elegantly showed that the type, density, and location of immune cells within human colorectal tumors predicts clinical outcome in terms of overall survival and progression free survival. Again, immunohistochemistry was used to stain CD3, CD8, Granzyme B and CD45RO. The authors however did not differentiate the results with regards to the role of any therapy, especially radiation- or chemotherapy. Additional previous work however clearly had shown the role of the adaptive immune response in colorectal cancer. This was published by Pages et al. (2005, New England Journal of Medicine, vol. 353: 2654-2666).
All the above mentioned publications state the use of numerous biological markers of the immune response in the course of understanding the mechanisms of the immune response against various cancers. However, these prior works provide no data relating to a statistical significant relationship between (i) the presence of, or the expression level of, these biological markers and (ii) treatment efficacy for chemotherapy, radiation therapy or immunotherapeutic interventions.
Although the previous publications shows good correlation between (i) the presence of, or the level of, some biological markers of the immune response and (ii) the effect on overall or progression free survival of cancers, the results of most of these prior studies also show that the use of the said biological markers were viewed exclusively as a confirmation of a prognostic cancer staging with conventional clinicopathological staging methods, or as an additional information to the said conventional cancer staging methods. For example, the biological marker used by Ishigami et al. (2002, above) was found to be useable exclusively with gastric carcinoma-bearing patients who were already diagnosed as “Stage IV” of the disease. Similarly, Zhang et al. (2003, above) concluded that prospective studies were needed to validate detection of intratumoral (CD3+) T cells in the classification and treatment of patients with ovarian carcinoma. Similarly, Diederichsen et al. (2003, above) disclosed the CD4+/CD8+ ratio as a biological marker having a survival prognostic value in colorectal cancer. However, these authors did not suggest that the said biological marker might be sufficient to influence a therapeutic decision or to predict therapeutic efficacy.
There is thus no report of reliable methods of cancer prognosis prediction that would make use exclusively of biological markers of the immune response from the host to estimate the efficacy of chemotherapy. Neither is there a reliable method for the prediction of treatment outcome in cancer patients.
Morris et al. (2008, Clin Cancer Res., vol 14: 1413-1417) used an analysis in the adjuvant treatment setting to state that tumor-infiltrating lymphocytes and perforation in colon cancer predict positive response to 5-fluorouracil chemotherapy. This however cannot be concluded from their data, because they only analysed the adjuvant treatment situation, so it remains unclear, how the adaptive immune response influences chemotherapy outcome in the light of only short term chemotherapy and long periods of follow-up.
Farmer et al. (2009, Nature Medicine, Vol 15: 68-74) identified a stroma-related gene signature that predicts resistance to neoadjuvant chemotherapy in breast cancer. They however did not identify immunological parameters for this prediction. In Baker et al. (U.S. Pat. No. 7,871,769, approved Apr. 19, 2011) the RNA expression levels of a set of genes is used to calculate a score to predict the response to chemotherapy but it does not measure the immune status of the patient.
Further, there is, today, no reliable marker available that would allow the prediction of the treatment outcome in all investigated cancer entities. This is also true for immunotherapeutic approaches, where also no good predictor of treatment outcome is available.
Notably, the availability of improved prediction methods would allow a better selection of patients for appropriate therapeutic treatments, especially in the situation of palliative treatment. Other important therapeutic interventions that could be improved by a better patient selection are immunotherapies. Immunotherapies are all therapies that either directly or indirectly modify the immune response or the immune system of a patient. For numerous cancers including colorectal cancers, the selection of an appropriate therapeutical treatment is purely empirical today. 55% of colorectal cancer patients undergoing palliative chemotherapy treatment have a response to chemotherapy. The rest of these patients only experiences side effects of the therapy. The genomic mutation status for KRAS is the only predictive marker for an antibody-based treatment regimen, as reported by Moroni et al. (2005, Lancet Oncol, vol 6: 279-86). Thus, the guided treatment would lead to better outcomes by reduction of toxicity and reduction of unnecessary side effects.